Design, Synthesis, and Evaluation of Lung-Retentive Prodrugs for Extending the Lung Tissue Retention of Inhaled Drugs

A major limitation of pulmonary delivery is that drugs can exhibit suboptimal pharmacokinetic profiles resulting from rapid elimination from the pulmonary tissue. This can lead to systemic side effects and a short duration of action. A series of dibasic dipeptides attached to the poorly lung-retentive muscarinic M3 receptor antagonist piperidin-4-yl 2-hydroxy-2,2-diphenylacetate (1) through a pH-sensitive-linking group have been evaluated. Extensive optimization resulted in 1-(((R)-2-((S)-2,6-diaminohexanamido)-3,3-dimethylbutanoyl)oxy)ethyl 4-(2-hydroxy-2,2-diphenylacetoxy)piperidine-1-carboxylate (23), which combined very good in vitro stability and very high rat lung binding. Compound 23 progressed to pharmacokinetic studies in rats, where, at 24 h post dosing in the rat lung, the total lung concentration of 23 was 31.2 μM. In addition, high levels of liberated drug 1 were still detected locally, demonstrating the benefit of this novel prodrug approach for increasing the apparent pharmacokinetic half-life of drugs in the lungs following pulmonary dosing.


■ INTRODUCTION
Drug inhalation has been successfully exploited as part of the management of respiratory diseases such as asthma and chronic obstructive pulmonary disease (COPD). 1,2 Recently, emerging literature evidence suggests that the pulmonary delivery route would also be beneficial for the treatment of cancer, 3 idiopathic pulmonary fibrosis (IPF), 4 respiratory infections, 5 and most recently, coronavirus disease . 6 However, a major limitation of local pulmonary delivery is that, typically, inhaled drugs exhibit a suboptimal pharmacokinetic profile characterized by a high maximum blood concentration (high C max ) that is achieved very shortly post administration (short T max ), resulting from the rapid elimination of compound from the pulmonary tissue. This can lead to systemic side effects and a short duration of action in the lungs. 7 Therefore, strategies to enhance lung residence time have been explored with the aim of improving the therapeutic index of inhaled therapies as well as decreasing their frequency of administration. 8,9 Due, in part, to a combination of mucociliary clearance and inherent high lung tissue permeability, achieving prolonged drug retention within the pulmonary tissue at a therapeutically acceptable concentration remains a major challenge. 7 Several strategies have evolved including (i) the diffusion microkinetic theory�where a high membrane partitioning of lipophilic bases into phospholipid bilayers explains the long duration of action of some bronchodilators; 10 (ii) receptor kinetics, in which slow receptor off-rates have been proposed as a hypothesis for the enhanced duration of action observed with both inhaled β 2 -agonists and muscarinic M3 receptor antagonists; 11 and (iii) reduction in solubility, where the slow dissolution of drug particles into the lung-lining fluid affords the potential for extended lung retention. 1 In addition, sustained-release formulations such as biodegradable polymer-based particles, 12,13 liposomes, 14 and poly(ethylene glycol)−drug ester conjugates 15 have all been assessed to increase drug residence time within the lung tissue. Over recent years, strategies to reduce pulmonary absorption by modifying the physicochemical properties of the therapeutic compound through drug design have resulted. At the forefront of these approaches was the observation that dibasic compounds per se have a very high capacity to exhibit long lung retention. 16−19 However, care needs to be applied in this strategy to ensure that there is sufficient local concentration of unbound drugs to have the required pharmacodynamic (PD) benefit. In addition, for intracellular targets, such as compounds designed to inhibit phosphatidylinositol 3-kinase (PI3K), a narrow physicochemical property window has been suggested for the balance of high lung tissue binding and cell permeation to enable the required long pharmacodynamics effect. 20 Taking into account the requirement to balance extended lung retention with a sustained concentration of the free drug, we report here on our initial work to evaluate a new prodrug approach for extended lung tissue retention. Its concept is based on attaching a known poorly lung tissueretentive compound to a lung tissue-retentive dibasic chemical substance through a pH-triggered release linker (slowly cleaved at pH > 6.5). The hypothesis is that the active drug would be slowly released in a controlled manner from the lung tissue depot, thus increasing the chances of successfully achieving "once-a-day" dosing regimens. Such a strategy could potentially be applied to inhaled drug classes acting on both cell surface and intracellular pharmacological targets as the active drug could freely be absorbed through cell membranes from its lung tissue depot after release to achieve the required extended duration of effect ( Figure 1).
To achieve our objective of developing a platform-based technology for inhaled lung delivery, the prodrug would need to: • Be readily synthesized with the concept applicable to a range of chemical classes • Be of low molecular weight to avoid long-term accumulation in the lung • Substantially increase the solubility of the parent drug to prevent local irritancy • Preferentially be pH-dependently cleaved (more stable at the pH of lung fluid (pH 6.5) and cleaved at the pH of blood (pH 7.4)). • Possess a nontoxic lung tissue-retentive moiety ("anchor") that can be easily excreted after activation.

■ RESULTS AND DISCUSSION
The prodrug design consisted of the parent drug bound to a lung-retentive moiety via a linker group, which bioactivates at a controlled rate to liberate the active drug molecule alongside nontoxic side products. To test the feasibility of this approach, Figure 1. Schematic representation of the novel lung-retentive prodrug concept. This would increase the drug half-life in the lung tissue and reduce its blood concentration; thus, the reduction would be released as acetaldehyde, while the lung tissue anchor would be eliminated by endogenous clearance mechanisms in the lungs, including absorption into the blood, mucociliary clearance, or uptake by macrophages.  we chose to functionalize the known muscarinic M 3 receptor antagonist 1, an active metabolite of enpiperate, 21−23 as 1 possesses the required synthetic handle that can be readily modified with our recently disclosed pH-sensitive linking group ( Figure 2). 24 In our initial studies, we explored the synthesis of a series of monobasic amino acids attached to 1 via a carbamate linker to explore the aqueous pH stability (pH 6.5) on both the positioning of the amine, its basicity (pK a ), and also the steric effect of substituents ( Figure 3).
The pK a of the amine governs the percentage ionized at the stated pH and is an important determinant in the hydrolytic cleavage of the carbamate by the anchimeric-assisted cleavage mechanism. The rate of cleavage would be pH-dependent as only the free amino compound could undertake the anchimeric-assisted delivery of water. NMR experiments in buffered pH 6.5 deuterated phosphate-buffered saline (PBS)/ D 6 -DMSO demonstrated the clean conversion of 17 to 1, arginine, and formaldehyde, which disproportionate to a mixture of acetic acid and ethanol (see Supporting Information S3) ( Figure 4). 24,25 The synthesized prodrugs were tested for their aqueous stability at pH 6.5 in an aqueous phosphate-buffered saline (PBS) ( Table 1).
The first parameter to be explored featured the position of the amine and how this might affect the rate of hydrolysis and the mechanism by which the prodrug cleaves. In the initial experiment, the glycine-derived prodrug 3 was shown to be readily hydrolyzed (T 1/2 = 0.52 h), whereas the β alaninederived prodrug 4 had increased stability (T 1/2 = 7.3 h). This trend in stability was not continued in the γand δ-substituted prodrugs (5 and 6, respectively), indicating a change in hydrolysis mechanism from anchimeric-assisted hydrolysis to an intramolecular nucleophilic cleavage mechanism, subsequently proven by NMR that showed the appearance of pyrrolidin-2-one and piperidin-2-one, respectively (n.b. no azetid-2-one was observed in the cleavage of 4). As expected, direct mono N-alkylation (7) reduced the pK a of the amine, which slightly increased the cleavage rate (compared to 3). Dialkylation (8), while again reducing the pK a , leads to a slower cleavage rate presumably for increased steric reasons and loss of H-bond donor properties. The use of proline (9) caused the cleavage rate to increase rapidly despite increasing the pK a ; this was rationalized due to Thorpe−Ingold effects, which push the two reactive components together to relieve steric strain. 26,27 The size of the amino acid side chain is another method by which the rate of prodrug hydrolysis can be controlled. By placing a large sterically hindering group next to the carbonyl group, an attack at this carbon would be sterically blocked and thus it was important to investigate how the size of the C-1 side chain affects its potential to sterically hinder hydrolysis. This data demonstrated how bulkier amino acid side chains slowed the hydrolysis of the prodrug by sterically hindering the anchimeric-assisted delivery of water. This resulted in slower cleavage rates in prodrugs (12−16) compared to that in 3.
From the monobasic prodrug cleavage data, it was clear that there were two possible approaches when designing dibasic amino acids. One route was to include an α-positioned amino acid with a large bulky side group to reduce the rate of cleavage, while the second approach involved a β-positioned amino acid, which would rely on the higher amine pK a to reduce the cleavage rate. Once incorporated, these αand βamino groups could then be derivatized to contain a second basic component, in which the cleavage mechanism would rely on anchimeric assistance ( Table 2).
The PBS buffer stability was determined to provide the maximum possible stability the prodrugs would have in the complete absence of enzymatic activity, i.e., the cleavage rate due to self-activation. This was tested in triplicate at 37°C at both pH 6.5 and 7.4. In all results, a clear propensity for cleavage at higher pH was observed, demonstrating the pHsensitive nature of the linking group. Compounds 17 and 20 displayed the same trend as was witnessed for the monobasic prodrugs, with the α-amino group giving a much faster cleavage rate than the β-amino prodrug. Unfortunately, as the mono-amino acid prodrugs (3−16) did not have a sufficient stability profile to progress further, an alternative strategy was sourced. To incorporate a sterically hindered α-positioned amino group, a series of dibasic dipeptides were synthesized. The sterically hindered ester group would then have increased resilience to nucleophilic attack, and thus the prodrug cleavage would depend less on the terminal amine pK a . This is because cleavage would now take place via an intramolecular diketopiperazine (DKP) formation mechanism, in which the intramolecular cyclization would occur via the nucleophilic attack of the primary amine of the first amino acid to the ester. This would create a 6-membered diketopiperazine, the formation of which would be more heavily influenced by steric hindrance than by the pK a of the nucleophilic amine. The rate of cleavage would be pH-dependent as only the free amino compound 23 could undertake the cyclization to the diketopiperazine. NMR experiments in buffered pH 6.5 deuterated PBS/D 6 -DMSO demonstrated the clean conversion to 1, substituted diketopiperazine, and formaldehyde, which disproportionate to a mixture of acetic acid and ethanol (see Supporting Information S2) ( Figure 5).
The prodrugs (21−28) were found to have a range of stabilities in phosphate buffer. As previously witnessed, the bulkier the C-1 side chain the lower the rate of cleavage, and hence the rate increases from compound 27, which has a relatively small α-substituent, to the highly hindered tertiary butyl group in compound 22. The most interesting results came from 23 and 26. To increase the chemical stability and reduce internal steric clashes, diketopiperazines place both groups in pseudo-equatorial positions resulting in a boat shape configuration ( Figure 6a). 28,29 For this reason, it was hypothesized that by inverting the stereochemistry of one of the amino acids in the dipeptide, a destabilizing steric clash would be formed, which would make the DKP less likely to form (Figure 6b), thus increasing the stability of the prodrug. However, when the stability of compounds 23 and 26 was tested, the prodrug stability in aqueous buffer did not improve relative to compounds 22 and 25. This could be explained by a shift in the DKP structure to a more chair-like conformation to reduce the steric clash between the hydrogen atom and the side chain and to balance the two carbonyl dipoles, which would be uneven in the boat conformation for the L,Ldipeptide ( Figure 6c).
Based on the phosphate stability studies and the prediction that enzymatic activity will increase the cleavage rate in biological media, the only compounds that proceeded to rat lung homogenate studies were compounds 20, 21−26, and 28. Han Wistar rat lung homogenate was prepared, and the pH (at a dilution of 1−4 in water) was experimentally determined to be 6.8. This meant that should our prodrugs not exhibit any enzymatic cleavage, the rate of pure intramolecular cleavage should be faster than in phosphate buffer at pH 6.5 but slower than at pH 7.4. The results demonstrated that most of the prodrugs were indeed subjected to a high level of enzymatic metabolism. The other clear conclusion to be drawn from the lung homogenate stability results was that compounds 23 and 26, in which the chirality of one of the amino acids was inverted from their natural isomer (22 and 25, respectively), were much more stable relative to their natural isomer matched pair. It is thought that by inverting the amino acid chirality, enzymatic cleavage at either the ester or amide bond will be reduced, possibly through the removal of the compound recognition for the enzymes' active site. To understand the enzymatic cleavage for compounds 22 and 23, the lung homogenate stability assay was repeated to detect the formation of the intermediate species 15, 18, and 1 produced if the terminal lysine residue was removed due to peptide amide bond hydrolysis ( Figure 7).
The results proved quite revealing with the natural amino acid analogue 22, clearly showing the intermediate 15, whereas 23 showed no evidence of intermediate 18, demonstrating that prodrug cleavage was occurring predominantly by a hydrolysis mechanism ( Figure 8).
Interestingly for 23, the rat lung homogenate stability and PBS stability were comparable (T 1/2 25.4 and 36.6 h, respectively), giving further evidence that the cleavage of 23 was mainly occurring through a nonenzymatic hydrolysis mechanism. Due to the chemical stability observed in rat lung homogenate for 23, it was possible to measure its rat lung homogenate binding. Compound 23 was 0.5% unbound compared to compound 1, which showed an unbound fraction of 18.3%. In addition, 23 had reasonable blood stability (T 1/2 7 h) compared to 22 (T 1/2 0.2 h). As a consequence of the encouraging in vitro data, the in vivo lung tissue retention capacity of 23 was evaluated in a rat intratracheal dosing pharmacokinetic (ITPK) study, where concentrations of compounds 23 and 1 would be measured in both rat lung and plasma after the intratracheal delivery of 23.
When designing the pharmacokinetic study, it was important to ensure that the dosing concentration was high enough to   allow for the detection of drug 1 and prodrug 23 at the final time point of 24 h. For this reason, the compounds were dosed at a relatively high concentration of 0.2 mg/kg and it was pleasing to note that 23 appeared as a clear solution in 5% EtOH in 95% PBS at pH 6.5. This demonstrates an immediate advantage of the dibasic prodrug system as drug insolubility can cause inflammation of the airway. After the parent muscarinic M3 receptor antagonist (1) was dosed via intratracheal administration (IT), blood samples were taken at 0.5, 1, and 3 h time points, and the concentration of drug was measured. In addition, the residual total lung concentration at 3 h was measured (Table 3).
Compound 1 could not be detected in blood as its plasma concentrations fell below the LLOQ for all time points. This was quite surprising as we know that drug 1 has reasonable plasma stability and so we might postulate that a combination   of hepatic and extrahepatic clearance mechanisms might be involved, as it is known that basic and dibasic compounds are susceptible to organo cation transporters (OCTs) 30 and it could be that the compounds are rapidly excreted into urine, as this is the case for some of the inhaled muscarinic antagonists as OCTs are expressed in the kidneys. At 3 h, the percentage of 1 remaining in the lung was calculated as 0.33% of the initial calculated total dose administered. This result demonstrates the very poor lung retention of compound 1, which would most likely be translated into a very short observed duration of action.
For the ITPK study of compound 23, plasma samples were then taken at 0.25, 0.5, 1, 2, 3, 5, 8, and 24 h time points, and the concentration of 1 and 23 was measured. At 24 h, the terminal total lung concentrations of 1 and 23 were measured ( Table 4).
As in the first study, compound 1 was not detected in any plasma samples and only a very low concentration of 23 was detected in the plasma up to 2 h post dosing. However, at 24 h, the total rat lung concentration was measured at 20,000 ng/mL (31.2 μM) for 23 (54% of the total dose delivered based on the calculated mass balance from the amount recovered in the lung tissue as a fraction of the total drug administered, assuming 100% lung deposition) and 361 ng/mL for the released active drug 1, which equates to an observed total lung concentration of 1.16 μM (an ∼27:1 ratio of 23 to 1 (3.7 ± 0.1%)). When working with prodrugs, much care is required in the interpretation of pharmacokinetic results as the prodrug could break down to release the parent drug during sample preparation. This is unlikely to be the case in this study as 23 has a half-life of 25.4 h when incubated at 37°C in the presence of rat lung homogenate, while sample preparation requires 10 min at an ambient temperature. However, to alleviate concerns around the prodrug stability in lung samples during sample preparation, a set of control experiments were conducted, where 23 in the IT dosing vehicle was spiked into rat lungs followed by their homogenization or directly into rat lung homogenates before samples were prepared for mass spectral quantification. There was very little evidence for the release of 1 from prodrug 23 during the sample workup. The initial fraction of 1 in the 23 stock solution was determined as ∼0.3−0.4%, whereas after sample preparation from lung homogenate or spiking into lungs, the percentage of 1 was quantified as 0.62 ± 0.03% (n = 3) and 0.56 ± 0.02% (n = 3), respectively. This would suggest that the conversion of 23 into 1 occurred within the lung tissue during the time course of the ITPK study and not during analytical sample preparation.

■ SYNTHESIS
The prodrugs (3−19) were synthesized through a common synthetic strategy (Scheme 1). Methyl benzilate was transesterified using a catalytic amount of sodium and N-Boc-4hydroxy piperidine to give after Boc-deprotection 1 isolated as the stable HCl salt. 31 Subsequent reaction of 1 with 1-(chloromethoxy)ethyl carbonochloridate affords the common precursor 2, which can be coupled with Boc-protected amino acids using silver(I) oxide and tetra-N-butylammonium bromide in toluene at 50°C 24 to give the Boc-protected compounds (3i−7i, 9i−18i), which were deprotected using anhydrous HCl in 1,4-dioxane to give (3−19) isolated as their mono-or dihydrochloride salts.

■ CONCLUSIONS
From consideration of the observation that dibasic compounds per se possess very good pharmacokinetic lung retention, a Compound 1 was dosed at 0.2 mg/kg IT in male rats (species: Crl Sprague Dawley, male, n = 3 per time point). Formulation of 5% ethanol in pH 6.5 PBS. <LLOQ = <lower limit of quantification, LLOQ = 25.0 ng/mL. The results shown are the mean of three replicates with standard deviation. Sample data analysis was performed at Sygnature Discovery, and the in-life phase was performed at Saretius Ltd. series of monobasic and dibasic prodrugs were synthesized and evaluated for their lung tissue binding and stability. Compound 23 was highlighted as a dibasic prodrug with the correct balance of measured lung tissue binding and chemical instability in PBS. The further evaluation suggested that the breakdown of prodrug 23 to active drug 1 occurred mainly through a pH-dependent diketopiperizine-forming cascade hydrolysis mechanism. The resulting prodrug 23 demonstrated high aqueous solubility and was dosed in a rat ITPK study to determine its pharmacokinetic profile. Quantification of plasma levels demonstrated very little systemic plasma exposure of 23 and released active drug 1 (1 was not detected at any time points). However, at 24 h post dose, a high total lung concentration of 23 was observed along with the released active drug 1. These initial results demonstrate a substantial increase in the lung residency of 1, when administered as the prodrug conjugate 23, when compared to dosing 1 alone (0.33% of a total administered dose observed at 3 h.). This result correlates with the observed increase in rat homogenate lung tissue binding between 1 (18.3% free) and 23 (0.5% free). The in vivo data supports an intrinsic pharmacokinetic benefit (by increasing concentrations of the active drug 1 and, more importantly, creating a reservoir of prodrug 23, potentially slow-releasing further active drug 1). It is our hypothesis that there should be an extended PD effect observed, as long as there is a sufficient local concentration of active drug 1 present. We appreciate that the safety implications of the extensive lung retention of 23 have not been fully investigated and that a finetuning to achieve a more desirable balance between lung tissue retention and duration of pharmacological action might be required. However, the attractiveness of the approach, in our opinion, lies precisely in the opportunity to select a prodrug with a tunable hydrolysis rate to achieve the required balance of prodrug lung retention and released active drug. Further work is ongoing within our laboratories to understand the lung tissue retention mechanisms of the dibasic prodrug moiety as well as further exploration of this new technology to other drug classes.
■ EXPERIMENTAL SECTION Chemistry: General Methods. Chemicals and solvents were provided by Fisher Scientific U.K., Acros Organics, Sigma-Aldrich, Merck Millipore, or Fluorochem. All reactions were monitored by TLC using Merck Silica Gel 60 Å F254 TLC plates or by LC−MS. Unless otherwise stated, all compounds were dried under high vacuum either at rt or within an oven at 40°C. LC−MS data was collected on a Shimadzu UFLCXR HPLC system coupled to an Applied Biosystems API 2000 LC/MS/MS electrospray ionization (ESI). The column used was a Phenomenex Gemini-NX 3 μm- 110AĈ 18, 50 × 2 mm at 40°C. The flow rate was 0.5 mL/min, and the UV detection was at 220 nm and 254 nm. Method 1 for the LC−MS ran for 1 min at 5% B; 5 to 98% B over 2 min, 98% B for 2 min, 98 to 5% B over 0.5 min, and then 5% for 1 min. Method 2 for the LC−MS ran for 1.5 min at 10% B; 10 to 98% B over 8 min; 98% B for 2 min; 98 to 10% B over 0.5 min, and then 10% B for 1 min, where solvent A is 0.1% formic acid in water and solvent B is acetonitrile. Unless otherwise stated, compounds reported had a purity >95% at the wavelength and method quoted. NMR spectroscopy was performed using a Bruker AV(III) HD 400 NMR spectrometer equipped with a 5 mm BBFO + probe, recording 1 H and 13 C NMR at 400.25 MHz and 100.66 MHz, respectively, or a Bruker AV(III) 500 NMR spectrometer equipped with a 5 mm dual 1H/13C helium-cooled cryoprobe, recording 1 H and 13 C NMR at 500.13 MHz and 125.77 MHz, respectively. NMR data was processed using iNMR (version 5.5.7) referencing spectra to residual solvents. Chemical shifts are  quoted as δ: values in ppm; coupling constants J = are given in Hz, and multiplicities are described as follows: s, singlet; d, doublet; t, triplet; q, quartet; qi, quintet; s, septet; m, multiplet; app, apparent; and bs, broad singlet.
All compounds submitted for in vitro evaluation had a purity >95% and in vivo >99%.
General Chemistry Procedure 1. To a solution of 2 (0.5 mmol, 1 equiv) in toluene (15 mL) were added the corresponding carboxylic acid (1.2 equiv), silver(I) oxide (1.2 equiv), and tetra-nbutylammonium bromide (0.2 equiv), and the reaction was heated at 65°C between 6 and 8 h. The reaction was cooled, diluted with ethyl acetate (15 mL), filtered, and concentrated. The resulting residue was purified by chromatography on silica gel.
General Chemistry Procedure 2. To a round-bottom flask was added the mono-or dihydrochloride salt as prepared in General Chemistry Procedure 1 or 3 (0.5 mmol, 1 equiv) in dry DCM (20 mL). To the suspension were added the corresponding protected amino acid (1.5 equiv), HATU (1.5 equiv), DMAP (0.5 equiv), and DIPEA (6 equiv). The resulting yellow solution was left to stir at room temperature for 6 h. The reaction was monitored by LC−MS, and once complete, the solution was diluted with DCM (50 mL) and washed with aqueous sodium hydrogen carbonate (3 × 25 mL). The organic layer was dried over anhydrous magnesium sulfate, filtered, and concentrated to give either the mono-or di-BOC-protected compounds, which were purified by chromatography to >99% purity.
General Chemistry Procedure 3. To a round-bottomed flask containing the Boc-protected prodrug obtained (0.5 mmol, 1 equiv) in dry dichloromethane (5 mL) was added trifluoroacetic acid (1 mL). The reaction was stirred at room temperature for 3 h and was concentrated. A solution of 2N HCl in diethyl ether (3 mL) was added to the residue, and the mixture was stirred for 15 min and concentrated, azeotroping with dry toluene (2 mL). The residue was triturated with a further aliquot of 2 N HCl in diethyl ether and concentrated to afford either the mono-or dihydrochloride salt.
Binding assays were determined in RED plates purchased from Thermofisher. Rat lung homogenate was either created on the day or stored at at least −20°C for a maximum of one freeze−thaw cycle. Whole rat blood was taken in-house and used on the day of assay, storing at 4°C if necessary. RED plates were prepared by placing the spiked matrix (100 μL, 1000 ng/mL) [rat lung homogenate] prepared in a t-vial into the first six RED ring chambers. The unspiked matrix (100 μL) was added to the final two RED chambers. Dialysis buffer (pH 6.5 phosphate-buffered saline, 300 μL) was added to the top six buffer chambers, and the bottom two buffer chambers remained empty to calculate recovery. The RED plate was sealed with a sealant tape and masking tape and incubated at 37°C for 4 h on an orbital shaker. Once the RED plate was prepared, the spiked matrix from the original t-vial (20 μL, 1000 ng/mL) was added to a labeled micronic immediately followed by control dialysis buffer (20 μL) and internal standard (300 μL, 6.25 ng/mL labetalol in MeCN and 17.5 ng/mL reserpine in MeCN), creating the time 0 sample. After 4 h, the RED plate was removed from the incubator, unsealed, and the spiked matrix from the original t-vial (20 μL, 1000 ng/mL) was added to a labeled micronic immediately followed by a control dialysis buffer (20 μL) and internal standard (300 μL, 6.25 ng/mL labetalol in MeCN and 17.5 ng/mL reserpine in MeCN), creating the time 240 sample. The RED plate was then sampled by removing 20 μL from each well into a labeled micronic tube. The incubated control matrix or incubated control PBS (20 μL) was added to the matrix match as follows: the incubated control PBS sample (20 μL) was added to the sample from the red ring (20 μL) in a labeled micronic tube in a 96well plate or the incubated control matrix (20 μL) was added to the buffer sample from buffer wells (20 μL) in a labeled micronic tube in a 96-well plate. All samples consist of matrix:PBS 1:1. To each micronic tube was added internal standard (300 μL, 6.25 ng/mL labetalol in MeCN and 17.5 ng/mL reserpine in MeCN), and the plate was shaken for 10 min and centrifuged for a further 20 min. The plate was then submitted for mass-spec analysis, quantifying the relative prodrug/drug mass ion peak against that of the internal standard.